Akt mediates insulin-stimulated phosphorylation of Ndrg2-: Evidence for cross-talk with protein kinase C θ
Akt mediates insulin-stimulated phosphorylation of Ndrg2-: Evidence for cross-talk with protein kinase C θ
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DOI：
10.1074/jbc.m401504200
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发表时间：
2004-04-30
影响因子：
4.8
通讯作者：
Schmitz-Peiffer, C
中科院分区：
文献类型：
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作者：
Burchfield, JG;Lennard, AJ;Schmitz-Peiffer, C
蛋白激酶Akt介导胰岛素的几种代谢和促有丝分裂作用，而蛋白激酶C(PKC)亚型的激活与抑制胰岛素的作用有关。我们之前已经证明PKCtheta和PKCepsilon在胰岛素抵抗高脂喂养的大鼠骨骼肌中都被激活，为了确定这些激酶的潜在底物，我们在体外将重组的PKC亚型与大鼠肌肉部分孵育。PKCtheta特异性地磷酸化了一个48 kDa的蛋白质，随后通过质谱仪鉴定为Ndrg2。Ndrg2与N-Myc下游调节蛋白1高度相关，后者与应激反应、细胞增殖和分化有关，尽管Ndrg2本身不受N-Myc抑制。Ndrg2含有几个潜在的磷酸化位点，包括三个Akt共有序列。在共表达PKCtheta或Akt的[P-32]正磷酸盐标记的C2C12肌肉细胞中，Ndrg2的磷酸化水平得到增强。用磷酸(丝氨酸/苏氨酸)Akt底物抗体进一步检测Ndrg2的磷酸化。胰岛素以Wortmannin和棕榈酸酯抑制的方式增加C2C12细胞中Ndrg2的磷酸化，而雷帕霉素、PD98059和双吲哚马来酰亚胺I没有作用，支持Akt的直接作用。Ndrg2的突变表明Thr-348是抗体检测到的主要磷酸化位点，Akt刺激该位点的磷酸化，而PKCtheta则使Ser-332磷酸化。然而，PKCtheta的过表达减弱了胰岛素对Thr-348磷酸化的影响，而不降低Akt的激活，这表明这是通过Ser-332处Ndrg2的磷酸化来调节的。我们的数据证实Ndrg2是一种新的胰岛素依赖的磷酸蛋白，并提示PKCtheta可能部分地通过减少Akt的磷酸化来抑制胰岛素的作用。
The protein kinase Akt mediates several metabolic and mitogenic effects of insulin, whereas activation of protein kinase C (PKC) isoforms has been implicated in the inhibition of insulin action. We have previously shown that both PKCtheta and PKCepsilon are activated in skeletal muscle of insulin-resistant high fat-fed rats, and to identify potential substrates for these kinases, we incubated recombinant PKC isoforms with rat muscle fractions in vitro. PKCtheta specifically phosphorylated a 48-kDa protein that was subsequently identified by mass spectrometry as Ndrg2. Ndrg2 is highly related to N-Myc downstream-regulated protein 1, which has been linked to stress responses, cell proliferation, and differentiation, although Ndrg2 itself is not repressed by N-Myc. Ndrg2 contains several potential phosphorylation sites, including three Akt consensus sequences. Ndrg2 phosphorylation was enhanced in [P-32] orthophosphate-labeled C2C12 muscle cells co-overexpressing either PKCtheta or Akt. Phosphorylation of Ndrg2 was examined further using a phospho (Ser/Thr) Akt substrate antibody. Insulin increased Ndrg2 phosphorylation in C2C12 cells in a wortmannin- and palmitate-inhibitable manner, whereas rapamycin, PD98059, and bisindoylmaleimide I had no effect, supporting a direct role for Akt. Mutation of Ndrg2 indicated that Thr-348 is the major phosphorylation site detected by the antibody and that Akt stimulates phosphorylation of this site, whereas PKCtheta phosphorylates Ser-332. PKCtheta overexpression, however, diminished the effect of insulin on Thr-348 phosphorylation without reducing Akt activation, suggesting that this is mediated through phosphorylation of Ndrg2 at Ser-332. Our data identify Ndrg2 as a novel insulin-dependent phosphoprotein and suggest that PKCtheta may inhibit insulin action in part by reducing its phosphorylation by Akt.